Streptococcus pneumoniae remains a leading cause of morbidity and mortality in community acquired respiratory infections. The third component of complement, C3, stands as the central mediator of host defense in susceptible patients who lack anti-capsular antibody. Over the past 5 years, we have identified two C3-degrading enzymes from S. pneumoniae: CppA, which degrades the C3 beta-chain; and PhpA, which cleaves the C3 alpha-chain into previously unrecognized fragments. Neither proteinase has any homolog in the database, and both are expressed by a wide variety of encapsulated clinical isolates. Intranasal immunization of mice with recombinant rCppA reduced nasopharyngeal colonization with a serotype 3 organism. Immunization of mice with rPhpA significantly reduced bacteremia and increased survival; in separate experiments, immunization with rPhpA was more effective than the serotype 3 conjugate vaccine in reducing nasopharyngeal colonization. In addition to the protective effects of CppA and PhpA in vivo, cppA- and phpA- mutants are more susceptible to C3-mediated opsonophagocytosis in vitro than is the isogenic parent. This revised proposal focuses on the mechanisms by which CppA and PhpA enable S. pneumoniae to elude C3-mediated killing in blood and lung. In Specific Aim One, we will characterize the mechanism of proteolysis by which CppA degrades the C3 beta-chain using chromogenic substrates and standard protease inhibitors. Truncation constructs expressed in Lactococcus lactis will be used to map the active site. A cppA- mutant in an encapsulated serotype 4 will be constructed. Specific Aim Two will focus on PhpA, a 79 kDa proteinase that cleaves the C3 alpha-chain into novel fragments of 97 and 83 kDa. Possible biologic activities of these C3 fragments in inhibiting C3 or neutrophils will be assayed. Biochemical techniques will be employed to understand how full-length PhpA liberates an internal 20 kDa polypeptide that appears to account for the majority of C3-cleaving activity. A phpA- mutant in an encapsulated serotype 4 will be constructed. Specific Aim Three will use a standard killing assay and a double mutant to test for additive or synergistic effects of CppA and PhpA. Other opsonins in blood (fibronectin) and lung (surfactant protein A) will be assessed as potential substrates for CppA and PhpA. Specific Aim Four will employ cppA- and phpA- mutants in the encapsulated strain to understand whether the effects of CppA and PhpA on C3-mediated killing contribute to virulence in a rabbit model of pneumonia and bacteremia. This revised proposal will define the role of two potent immunogens in pneumococcal pathogenesis.